No Evidence of False-negative P. Falciparum Rapid Diagnostic Results in Monrovia, Liberia

Abstract Background: Malaria diagnosis relies mainly on the use of rapid diagnostic tests (RDTs). The majority of commercial RDTs used in Africa detect the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). pfhrp2/3 gene deletions can therefore lead to false-negative RDT results. This study aimed to evaluate the frequency of PCR-confirmed, false-negative P. falciparum RDT results in Monrovia, Liberia.Methods: We used PfHRP2-based RDT (Paracheck Pf®) and microscopy results from 1038 individuals with fever or history of fever (n=951) and pregnant women at first antenatal care (ANC) visit (n=87) enrolled in the Saint Joseph Catholic Hospital (Monrovia) from March to July 2019 to assess the frequency of false-negative RDT results. True false negatives were confirmed by detecting the presence of P. falciparum DNA by quantitative PCR in samples from individuals with discrepant RDT and microscopy resultsResults: One hundred and eighty-six (19.6%) and 200 (21.0%) of the 951 febrile participants had a P. falciparum positive result by RDT and microscopy, respectively. Positivity rate increased with age and the reporting of joint pain, chills and shivers, vomiting and weakness, and increased with the presence of coughs and nausea. The positivity rate at first ANC visit was 5.7% (n=5) and 8% (n=7) by RDT and microscopy, respectively. Out of 207 Plasmodium infections detected by microscopy, 22 (11%) were negative by RDT. qPCR confirmed absence of P. falciparum DNA in the sixteen RDT-negative but microscopy-positive samples which were available for molecular testing. Conclusion: There is no qPCR-confirmed evidence of false-negative RDT results due to pfhrp2/pfhrp3 deletions in this study conducted in Monrovia (Liberia). This indicates the appropriate performance of PfHRP2-based RDTs for the diagnosis of malaria in Liberia. Nevertheless, active surveillance for the emergence of PfHRP2 deletions is required.

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No evidence of false-negative Plasmodium falciparum rapid diagnostic results in Monrovia, Liberia

Malaria Journal ◽

10.1186/s12936-021-03774-3 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Mandella King ◽

Alexander E. George ◽

Pau Cisteró ◽

Christine K. Tarr-Attia ◽

Beatriz Arregui ◽

...

Keyword(s):

Plasmodium Falciparum ◽

False Negative ◽

Malaria Diagnosis ◽

Rapid Diagnostic Tests ◽

Molecular Testing ◽

False Negatives ◽

Gene Deletions ◽

History Of ◽

Catholic Hospital ◽

Or History

Abstract Background Malaria diagnosis in many malaria-endemic countries relies mainly on the use of rapid diagnostic tests (RDTs). The majority of commercial RDTs used in Africa detect the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). pfhrp2/3 gene deletions can therefore lead to false-negative RDT results. This study aimed to evaluate the frequency of PCR-confirmed, false-negative P. falciparum RDT results in Monrovia, Liberia. Methods PfHRP2-based RDT (Paracheck Pf®) and microscopy results from 1038 individuals with fever or history of fever (n = 951) and pregnant women at first antenatal care (ANC) visit (n = 87) enrolled in the Saint Joseph’s Catholic Hospital (Monrovia) from March to July 2019 were used to assess the frequency of false-negative RDT results. True–false negatives were confirmed by detecting the presence of P. falciparum DNA by quantitative PCR in samples from individuals with discrepant RDT and microscopy results. Samples that were positive by 18S rRNA qPCR but negative by PfHRP2-RDT were subjected to multiplex qPCR assay for detection of pfhrp2 and pfhrp3. Results One-hundred and eighty-six (19.6%) and 200 (21.0%) of the 951 febrile participants had a P. falciparum-positive result by RDT and microscopy, respectively. Positivity rate increased with age and the reporting of joint pain, chills and shivers, vomiting and weakness, and decreased with the presence of coughs and nausea. The positivity rate at first ANC visit was 5.7% (n = 5) and 8% (n = 7) by RDT and microscopy, respectively. Out of 207 Plasmodium infections detected by microscopy, 22 (11%) were negative by RDT. qPCR confirmed absence of P. falciparum DNA in the 16 RDT-negative but microscopy-positive samples which were available for molecular testing. Among the 14 samples that were positive by qPCR but negative by RDT and microscopy, 3 only amplified pfldh, and among these 3 all were positive for pfhrp2 and pfhrp3. Conclusion There is no qPCR-confirmed evidence of false-negative RDT results due to pfhrp2/pfhrp3 deletions in this study conducted in Monrovia (Liberia). This indicates that these deletions are not expected to affect the performance of PfHRP2-based RDTs for the diagnosis of malaria in Liberia. Nevertheless, active surveillance for the emergence of PfHRP2 deletions is required.

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Deletions of pfhrp2 and pfhrp3 genes in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda

10.21203/rs.3.rs-42855/v1 ◽

2020 ◽

Author(s):

Sam L Nsobya ◽

Andrew Walakira ◽

Elizabeth Namirembe ◽

Moses Kiggundu ◽

Joaniter I Nankabirwa ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Protein Expression ◽

False Negative ◽

Plasmodium Species ◽

Pcr Amplification ◽

Rapid Diagnostic Tests ◽

False Negatives ◽

Cross Sectional ◽

True Prevalence ◽

Blood Smears

Abstract Background: Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the homologous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on protein expression, and our understanding of the true prevalence of deletions is incomplete. Methods: We investigated pfhrp2 / pfhrp3 deletions in blood samples from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. We evaluated samples with positive Giemsa-stained thick blood smears and negative PfHRP2-based RDTs by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression ofPfHRP2. Results: Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum was identified in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum -positive samples there was a failure to amplify pfhrp2 or pfhrp3 :in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum -positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay. Conclusion: False negative RDTs were uncommon, and deletions in pfhrp2 and pfhrp3 explained some of these findings, although most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

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PO 8271 PFHRP2 GENE DELETIONS IN PLASMODIUM FALCIPARUM AND SCHISTOSOMA MANSONI CO-INFECTIONS: AN EMERGING CHALLENGE FOR MALARIA RAPID DIAGNOSTIC TESTS

BMJ Global Health ◽

10.1136/bmjgh-2019-edc.64 ◽

2019 ◽

Vol 4 (Suppl 3) ◽

pp. A25.2-A25

Author(s):

Hilda Echelibe ◽

Masumbe Netongo Palmer ◽

Nji Akindeh ◽

Wilfred Mbacham

Keyword(s):

Plasmodium Falciparum ◽

Schistosoma Mansoni ◽

Diagnostic Tests ◽

False Negative ◽

Rapid Diagnostic Tests ◽

Sub Saharan Africa ◽

P Value ◽

Gene Deletions ◽

Malaria Rdts ◽

Malaria Rapid Diagnostic Tests

BackgroundMalaria and schistosomiasis are infections that have a great impact in sub-Saharan Africa based on their high morbidity and mortality rates. We suggest the possibility that the microenvironment created from interactions between the parasites involved generates a pressure on the malaria parasite which could in turn favour the parasite’s adaptation or escape through Pfhrp2 gene deletions. Thus, this study aimed at determining the association between the co-infection with both parasites and false-negative PfHRP2-based malaria rapid diagnostic tests which occur because of these deletions.MethodsThis pilot study was conducted in a total of 149 children aged 7–17 years living in Yorro, located in the Mbam-Inoubou division of the Center region of Cameroon. We collected fresh stool samples from each participant to identify Schistosoma mansoni (Sm) eggs by Kato Katz method and blood samples to identify the ring stages of Plasmodium falciparum (Pf) by thick smear. Malaria rapid diagnostic test and Pfhrp2 gene polymerase chain reaction were performed. The association between the co-infection with Sm/Pf and the false-negative malaria RDTs was determined by the Fisher’s exact test. A p value<0.05 was considered statistically significant.ResultsOur results showed that samples were singly infected with Sm, Pf, co-infected (Sm/Pf) and negative for both infections at frequencies of 12%, 43%, 30.2% and 14.8% respectively. False-negative PfHRP2-based RDTs were observed in 4.7% of the participants. A higher frequency (5/7) of the cases with false-negative malaria RDTs were co-infected with Sm/Pf. A p value of 0.027 showed statistical significance in the association of Sm/Pf co-infection and false-negative PfHRP2-based RDTs.ConclusionA significant association of Plasmodium falciparum and Schistosoma mansoni co-infection with false-negative PfHRP2-based RDTs supports the case for a plausible implication of Pfhrp2 gene deletions, with consequences for malaria rapid diagnostic testing.

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Prozone-like phenomenon in travellers with fatal malaria: report of two cases

The Journal of Infection in Developing Countries ◽

10.3855/jidc.5454 ◽

2015 ◽

Vol 9 (03) ◽

pp. 321-324 ◽

Cited By ~ 6

Author(s):

Lurdes Santos ◽

Nuno Rocha Pereira ◽

Paulo Andrade ◽

Paulo Figueiredo Dias ◽

Carlos Lima Alves ◽

...

Keyword(s):

Diagnostic Tests ◽

False Negative ◽

Specific Antigen ◽

Malaria Diagnosis ◽

High Sensitivity ◽

Rapid Diagnostic Tests ◽

Microscopy Observation ◽

Negative Results ◽

False Negative Results ◽

Potential Benefits

Malaria diagnosis remains a concern in non-endemic countries, with rapid diagnosis being crucial to improve patients’ outcome. Rapid diagnostic tests have high sensitivity but they also have flaws and false-negative results that might jeopardize malaria diagnosis. Some false-negative results might relate to a prozone-like effect. The authors describe two patients with false-negative rapid diagnostic tests in which a prozone-like effect might have been involved. The authors highlight that these tests should not be used without accompanying light microscopy observation of blood films and discuss potential benefits of using rapid diagnostic tests with more than one specific antigen for Plasmodium falciparum.

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Impact of seasonal variations in Plasmodium falciparum malaria transmission on the surveillance of pfhrp2 gene deletions

eLife ◽

10.7554/elife.40339 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 6

Author(s):

Oliver John Watson ◽

Robert Verity ◽

Azra C Ghani ◽

Tini Garske ◽

Jane Cunningham ◽

...

Keyword(s):

False Negative ◽

Plasmodium Falciparum Malaria ◽

Rapid Diagnostic Tests ◽

World Health ◽

Seasonal Fluctuations ◽

Gene Deletions ◽

Control Programmes ◽

Sampling Intervals ◽

Optimum Sampling ◽

Health Organization

Ten countries have reported pfhrp2/pfhrp3 gene deletions since the first observation of pfhrp2-deleted parasites in 2012. In a previous study (Watson et al., 2017), we characterised the drivers selecting for pfhrp2/3 deletions and mapped the regions in Africa with the greatest selection pressure. In February 2018, the World Health Organization issued guidance on investigating suspected false-negative rapid diagnostic tests (RDTs) due to pfhrp2/3 deletions. However, no guidance is provided regarding the timing of investigations. Failure to consider seasonal variation could cause premature decisions to switch to alternative RDTs. In response, we have extended our methods and predict that the prevalence of false-negative RDTs due to pfhrp2/3 deletions is highest when sampling from younger individuals during the beginning of the rainy season. We conclude by producing a map of the regions impacted by seasonal fluctuations in pfhrp2/3 deletions and a database identifying optimum sampling intervals to support malaria control programmes.

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Prevalence of Plasmodium falciparum Isolates Lacking the Histidine Rich Protein 2 Gene Among Symptomatic Malaria Patients in Kwilu Province, DR. Congo.

10.21203/rs.3.rs-79327/v1 ◽

2020 ◽

Author(s):

Yannick Bazitama Munyeku ◽

Alain Abera Musaka ◽

Medard Ernest ◽

Chris Smith ◽

Paul Mankadi Mansiangi ◽

...

Keyword(s):

Gene Deletion ◽

Statistical Significance ◽

False Negative ◽

Secondary Data ◽

Malaria Diagnosis ◽

Malaria Prevention ◽

Dr Congo ◽

Cross Sectional ◽

Gene Deletions ◽

Symptomatic Malaria

Abstract BackgroundMalaria rapid diagnostic tests have become a primary and critical tool for malaria diagnosis in malaria-endemic countries where PfHRP2-based RDTs are widely used. However, in the last decade, the accuracy of PfHRP2-based RDTs has been challenged by the emergence of P. falciparum strains harbouring deletions of the pfhrp2 gene, resulting in false-negative results. In DR. Congo, little is known about the prevalence of the pfhrp2 gene deletion among P. falciparum isolates infecting symptomatic patients, especially in low to moderate transmission areas where pfhrp2 deletion parasites are assumed to emerge and spread. Here we determine the local prevalence and factors associated with pfhrp2 gene deletions among symptomatic malaria patients in the Kwilu province of the Democratic Republic of Congo, a low to moderate malaria transmission area.MethodsWe used secondary data from a prospective health facility-based cross-sectional study conducted on 684 individuals of all ages, seeking healthcare with symptoms suggestive of malaria from October to December 2018 in 34 randomly selected health facilities. Sociodemographic, malaria prevention and treatment practices, and clinical variables were collected using a pre-tested structured questionnaire. Patients’ medical records were used to collect additional clinical data. Blood was collected for microscopy, PfHRP2-RDT, and spotted onto Whatman filter paper for downstream genetic analysis. Genomic DNA was extracted and used to perform PCR assays for the detection and confirmation of pfhrp2 gene deletions. Data were entered and analysed using STATA15. Fischer’s exact and the Kruskal-Wallis tests were applied to look for associations between exposures and the pfhrp2 gene deletion with a level of statistical significance set at p < 0.05. ResultsThe overall prevalence of the pfhrp2 gene deletion was 9.2% (95% CI 6.7% – 12.1%). The deletion of the pfhrp2 gene was associated with health zone of origin (p=0.012) and age (p=0.019). Among false-negative PfHRP2-RDT results, only 9.9% were due to pfhrp2 gene deletion.ConclusionP. falciparum isolates with pfhrp2 gene deletions are relatively common among symptomatic patients in Kwilu province. Further investigations are needed to provide enough evidence for policy change. Meanwhile, the use of RDTs targeting PfHRP2 and pLDH antigens could limit the spread of deleted isolates.

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Modelling the drivers of the spread of Plasmodium falciparum hrp2 gene deletions in sub-Saharan Africa

eLife ◽

10.7554/elife.25008 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 33

Author(s):

Oliver J Watson ◽

Hannah C Slater ◽

Robert Verity ◽

Jonathan B Parr ◽

Melchior K Mwandagalirwa ◽

...

Keyword(s):

Democratic Republic Of Congo ◽

False Negative ◽

Malaria Diagnosis ◽

Parasite Prevalence ◽

Malaria Prevalence ◽

Sub Saharan Africa ◽

Gene Deletions ◽

High Frequencies ◽

Sub Saharan ◽

Selection Of

Rapid diagnostic tests (RDTs) have transformed malaria diagnosis. The most prevalent P. falciparum RDTs detect histidine-rich protein 2 (PfHRP2). However, pfhrp2 gene deletions yielding false-negative RDTs, first reported in South America in 2010, have been confirmed in Africa and Asia. We developed a mathematical model to explore the potential for RDT-led diagnosis to drive selection of pfhrp2-deleted parasites. Low malaria prevalence and high frequencies of people seeking treatment resulted in the greatest selection pressure. Calibrating our model against confirmed pfhrp2-deletions in the Democratic Republic of Congo, we estimate a starting frequency of 6% pfhrp2-deletion prior to RDT introduction. Furthermore, the patterns observed necessitate a degree of selection driven by the introduction of PfHRP2-based RDT-guided treatment. Combining this with parasite prevalence and treatment coverage estimates, we map the model-predicted spread of pfhrp2-deletion, and identify the geographic regions in which surveillance for pfhrp2-deletion should be prioritised.

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Deletions of pfhrp2 and pfhrp3 genes were uncommon in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda

Malaria Journal ◽

10.1186/s12936-020-03547-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Sam L. Nsobya ◽

Andrew Walakira ◽

Elizabeth Namirembe ◽

Moses Kiggundu ◽

Joaniter I. Nankabirwa ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Protein Expression ◽

Diagnostic Tests ◽

False Negative ◽

Pcr Amplification ◽

Rapid Diagnostic Tests ◽

False Negatives ◽

Cross Sectional ◽

True Prevalence ◽

Blood Smears

Abstract Background Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the paralogous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum infections from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on PfHRP2 protein expression, and understanding of the true prevalence of deletions is incomplete. Methods Deletions of pfhrp2/pfhrp3 in blood samples were investigated from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. Samples with positive Giemsa-stained thick blood smears, but negative PfHRP2-based RDTs were evaluated by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression of PfHRP2. Results Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum DNA was identified by PCR in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum-positive samples there was a failure to amplify pfhrp2 or pfhrp3: in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum-positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay. Conclusion False negative RDTs were uncommon. Deletions in pfhrp2 and pfhrp3 explained some of these false negatives, but most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

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Deletions of pfhrp2 and pfhrp3 genes were uncommon in rapid diagnostic test-negative Plasmodium falciparum isolates from Uganda

10.21203/rs.3.rs-42855/v2 ◽

2020 ◽

Author(s):

Sam L Nsobya ◽

Andrew Walakira ◽

Elizabeth Namirembe ◽

Moses Kiggundu ◽

Joaniter I Nankabirwa ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Protein Expression ◽

Diagnostic Tests ◽

False Negative ◽

Pcr Amplification ◽

Rapid Diagnostic Tests ◽

False Negatives ◽

Cross Sectional ◽

True Prevalence ◽

Blood Smears

Abstract BackgroundRapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the paralogous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum infections from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on PfHRP2 protein expression, and understanding of the true prevalence of deletions is incomplete.MethodsDeletions of pfhrp2/pfhrp3 in blood samples were investigated from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. Samples with positive Giemsa-stained thick blood smears, but negative PfHRP2-based RDTs were evaluated by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression of PfHRP2.ResultsOf 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum DNA was identified by PCR in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum-positive samples there was a failure to amplify pfhrp2 or pfhrp3: in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum-positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay.ConclusionFalse negative RDTs were uncommon. Deletions in pfhrp2 and pfhrp3 explained some of these false negatives, but most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.

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